Integrins are non-covalently linked α/β heterodimeric receptors that mediate cell adhesion, migration and signaling. Together with their ligands, integrins play central roles in many processes including development, hemostasis, inflammation and immunity, and in pathologic conditions such as cancer invasion and cardiovascular disease. The β2 integrins, which have a common β-subunit (β2, CD18) but distinct α-subunits (CD11a, CD11b, CD11c and CD11d), are critical leukocyte receptors that are important not only for the function of leukocytes but also the development of the inflammatory response in vivo. Leukocytes normally circulate in the vasculature in a quiescent state, but in response to inflammatory stimuli, adhere, transmigrate across the vascular endothelium, and enter areas of tissue inflammation where they participate in the destruction and removal of infectious agents and in amplifying the process of inflammation. The integrin CD11b/CD18 (complement receptor type 3 (CR3), Mac-1 or αMβ2) is the predominant β2 integrin receptor in neutrophils, macrophages and monocytes and mediates a large number of pro-inflammatory functions in these cells. CD11b/CD18 recognizes a wide variety of ligands, including the complement fragment iC3b, fibrinogen, blood-clotting factor X, CD54 (ICAM-1), the hookworm neutrophil inhibitory factor (NIF), and denatured proteins such as bovine serum albumin (BSA). Studies in CD11b−/− mice have shown that this integrin has a distinct and cooperative role (with integrin CD11a) in the inflammatory process. In addition to the knockout mice studies, the biological importance of this integrin in maintaining immunological homeostasis has also been illustrated by different pathological conditions where integrins are absent or defective—loss of functional β2 integrins causes life-threatening infections in humans and mutations result in leukocyte adhesion deficiency type 1, where circulating neutrophils fail to adhere to or migrate across the endothelium and the patients are susceptible to recurrent, life-threatening bacterial infections. Similarly, improper excessive activation of leukocyte integrins is also harmful, as over-activation of β2 integrins contributes to sustained inflammation, ischemia-reperfusion injury (including acute renal failure, atherosclerosis and autoimmune disorders, tissue damage) and the development of various autoimmune diseases. CD11b/CD18 is also implicated in stroke, neointimal thickening in response to vascular injury2, bullous pemphigoid, and neonatal obstructive nephropathy. Thus, there is a considerable potential for agents that block the binding of CD11b/CD18 to its physiologic ligands as therapeutics for the treatment of such inflammatory conditions.
Physiologic ligand binding by CD11b/CD is divalent-cation dependent and is mediated by CD11b von Willebrand factor type A (VWFA) domain, CD11bA-domain (A-domain). Blocking anti-CD11b/CD18 antibodies decreases ischemia/reperfusion injury, the area of myocardial infarction and liver cell injuries, and diminishes neointimal thickening and restenosis after balloon injury of carotid arteries in animal models. These antibodies are also effective in the treatment of endotoxic challenge and hemorrhagic shock and autoimmune injury in various organs including the kidney. However, antibody therapy is not ideal because adverse effects due to nonselective blockade of various other leukocyte functions may lead to severe complications. Similarly, neutrophil inhibitory factor (NIF), a 41-kDa glycoprotein ligand-mimic, is effective in attenuating the deleterious effects of excessive neutrophil activation in animal models, but its large size and immunogenicity preclude its use as a therapeutic agent. Additionally, although blockade of the binding sites of integrins with ligand-mimetic peptides or small molecules has proven effective in inhibiting the activities of β1 and β3 integrins, peptides derived either from CD11b/CD18 ligands or anti-CD11b/CD18 antibodies were not very efficacious in blocking ligand binding in vitro. The failure of these ligand-mimetic peptides to block the interaction between iC3b and CD11b/CD18 may be due to their improper conformation in solution or to the size of the ligand binding sites, which may be too extensive to block with a small peptide.
Current assays for the identification of regulators of CD11b/CD18 rely on purified proteins adsorbed to microtiter plates. Even though these assays are compatible with high-throughput screening (HTS), purification of the requisite amount of CD11b/CD18 from mammalian cells for a HTS campaign can be exceedingly difficult and the natural conformation of integrin may not be retained upon adsorption to the plastic surfaces. Optimized cell-based phenotypic assays that can be readily utilized in an HTS environment for the rapid identification of small molecule regulators of this important integrin are currently lacking. Recent reports also suggest that CD11b/CD18 has a central role in the resolution of inflammatory processes by modulating the egress of adherent neutrophils from the site of inflammation. This suggests that small molecule agonists of CD11b/CD18 may also have a role in treatment of certain inflammatory and other conditions. Here again, simple cell-based phenotypic assays for ready HTS adaptation are currently lacking.
Therefore what is needed are small molecules that selectively modulate CD11b/CD18 ligand binding, especially by targeting allosteric regulatory sites, such as the hydrophobic site-for-isoleucine (SILEN) pocket in CD11b/CD1824, which may prove to be a more promising therapeutic strategy. An effective assay for the rapid identification of small molecule modulators of integrin CD11b/CD18 is also needed.